This proposal's objective is to gain a better understanding of the molecular mechanisms underlying the development of tolerance of opiates. The findings of this study may contribute to the design of therapeutic agents that will provide analgesia comparable to that of morphine without inducing tolerance. These experiments will examine changes that occur in the mu receptor-signaling pathway in cultured cells. Additionally, they will examine changes that occur in the cellular localization of RGS4 in response to chronic mu agonist stimulation. The specific aims are designed to answer the following: 1. Are RGS4 and the mu opioid receptor co-localized in the rat brain. Does chronic agonist treatment causes RGS4 translocation from the nucleus to the cytosol? 2. How does chronic DAMGO treatment enhances the efficacy of RGS4 in blunting mu opioid receptor mediated inhibition of adenylyl cyclase activity? The experiments are designed to test the following hypothesis: Chronic activation of the mu receptor causes RGS4 to translocate from the nucleus to the cytosol. Also, chronic mu receptor activation causes RGS4 to associate with the receptor, bringing RGS4 in close proximity to the receptor-associated Gi-type G proteins, resulting in an increase in the GTPase of these G proteins.